Hypertension is a cardiovascular risk factor that can lead to ischemic injury, myocardial infarction (MI) and heart failure. During these diseases, the heart undergoes hypertrophy and fibrosis. These processes are stimulated by release of cytokines (the pro-inflammatory cytokine interleukin-1 beta, IL), neurohormonal agents (beta-adrenergic hormones) and vasoactive hormones with trophic properties, such as endothelin-1 (ET) and angiotensin II (Ang II). These agents help regulate genes involved in the inflammatory response, including cyclooxygenase-2 (COX-2), resulting in prostanoid (PGE2, PGF2 alpha, PGI 2) production by cardiac myocytes. In fact, myocyte PGE 2 levels are increased dramatically compared with other prostanoids (PGs), as PGE2 synthase is induced along with COX-2. Preliminary data show that COX-2 mRNA is induced in the mouse heart following MI, and 2 wk treatment with NS-398, a specific inhibitor, reduces collagen content. Our in vitro data indicate that PGs, including PGF alpha, PGE2 and the PGE2 analogue sulprostone, induce hypertrophic growth of myocytes, fibroblast proliferation and potentiate IL induction of COX-2. The effect of PGF2 alpha is mediated by binding to the FP receptor, while there are four EP receptor subtypes that can mediate PGE2?s effect. Our preliminary data indicate that EP1 and EP3 are likely involved in the trophic effects of PGE2. In contrast, the transcription factor PPAR gamma opposes IL stimulation of PGE2 production, as well as myocyte hypertrophy. We will study the contribution of COX-2 products to hypertrophy and fibrosis in a mouse model of MI. We hypothesize that induction of COX-2 and elaboration of inflammatory prostanoids, acting locally through myocyte and fibroblast EP1/EP3 and FP receptors, will result in hypertrophy and fibrosis (proliferation of fibroblasts and collagen deposition) following MI, which can be reduced by either a COX-2 inhibitor or a PPAR gamma agonist. In Aim 1 we will examine the regulation of COX-2 and PGE2 synthase in myocytes, and determine whether these proteins co-localize to the same intracellular compartment in a perinuclear distribution and whether PGE2 exerts intracrine effects when combined with nuclear EP1 and/or EP3. In Aim II, we will study upregulation of expression and binding of the EP1/EP3 and FP receptors in myocytes and fibroblasts in vitro by trophic factors and the inflammatory cytokine IL-1 and in vivo by chronic ischemia (post-MI). In Aim 3, we will use protein synthesis and BNP gene expression in vitro as markers to examine the signaling mechanisms by which PGE2 and PGF2 alpha regulate hypertrophy in myocytes, focusing on cross-coupling of G alpha i to Src and subsequent activation of PI3-kinase, p42/44 MAPK, and p70s6k. In Aim 4, we will examine how PGE2 and PGF2 alpha increase transit of fibroblasts through the cell cycle using Western blot of cell-cycle regulatory proteins (cyclins and cyclindependent kinases). We will also study the signaling mechanisms by which PGE2 and PGF alpha regulate collagen synthesis. In Aims III and IV we will verify our in vitro data using a mouse MI model to test how inhibition of COX-2 or deletion of EP3 prevents the development of hypertrophy and fibrosis. In addition, a molecular model of COX-2 inhibition will be developed, whereby COX-2 antisense is induced only in the mouse heart using the tetracycline-off (TET-off) system. Finally, in Aim 5 we will study the effect of PPAR gamma agonists on expression of COX-2 and PGE2 synthase and activation of transcription factors that are involved in growth (NF kappa beta and AP-1). The contribution of COX-2 products to cardiac pathophysiology is virtually unexplored. Our studies will use an integrative approach to study the cellular and molecular basis for the deleterious effects of the inflammatory protein COX-2 and EP1/3 and FP in a model of chronic ischemia characterized by hypertrophy and fibrosis.